Development of cancer involves aberrant control of cellular proliferation due to activation of oncogenes as well as inactivation of tumor suppressors, which protect against unregulated growth by inducing apoptosis or growth arrest (senescence) of pre-malignant cells. Ras proto-oncogenes are often mutationally activated in cancer cells, while the p53 or RB tumor suppressor pathways are almost universally disabled. Detailed knowledge of oncogenic and anti-oncogenic pathways is essential for understanding how cancers develop and to identify unique vulnerabilities of tumor cells that can be exploited for therapeutic intervention. Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) transcription factors, especially their involvement in regulating cell proliferation and tumorigenesis. Our research focuses primarily on C/EBPbeta and its role as a downstream effector of oncogenic Ras signaling. Tumor studies using Cebpb null mice as well as analysis of C/EBPb-deficient cells revealed that C/EBPb has pro-oncogenic functions. However, in RasV12-expressing fibroblasts (MEFs) C/EBPb is also required for oncogene-induced senescence (OIS), an intrinsic tumor suppressive mechanism. In the latter context C/EBPb acts to arrest cellular proliferation by a pathway requiring RB:E2F. Thus, C/EBPb possesses both pro- and anti-tumorigenic activities. Because it plays an important role in cellular responses to Ras, we wish to elucidate the mechanisms by which C/EBPb expression and activity are controlled by oncogenic and physiological Ras signaling and thus to better understand the dual activities of C/EBPb in suppressing or promoting cancer in vivo. Posttranslational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed protein whose activity can be stimulated by oncogenic Ras or growth factor signals. C/EBPb auto-inhibition involves three regions in the N-terminal half of the protein that, together with sequences at the C terminus, are predicted to form a hydrophobic core. The folded core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb activation by Ras signaling involves several inducible posttranslational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper as an important regulator of C/EBPb DNA-binding and homodimerization. Recently, we identified two Ras-induced phosphorylation sites and a methylated arginine within an auto-inhibitory domain that are required for functional of C/EBPb activation by Ras. Ongoing work includes identifying the effector pathways and modifying enzymes downstream of Ras that regulate these PTMs and understanding their roles in controlling the biological and oncogenic functions of C/EBPb. Pro-oncogenic role of C/EBPb: Previous studies showed that C/EBPb-deficient mice are resistant to development of carcinogen-induced skin tumors. We have extended these findings by using other carcinogenesis protocols and mouse tumor models. C/EBPb knockout mice exposed to ENU exhibit impaired lymphomagenesis and reduced incidence/malignancy of a broad spectrum of other cancers compared to WT animals. Also, growth of mouse colon carcinoma cells is less efficient in C/EBPb KO mice than in WT animals, possibly due to decreased circulating levels of IGF-1 in the mutant mice. C/EBPb null mice also develop fewer tumors than WT mice in a K-ras lung carcinogenesis model. Future studies will address the possible involvement of proinflammatory cytokines (IL-6), chemokines (IL-8), and their receptors as oncogenic targets of C/EBPb in Ras-induced and other cancers. Tumor suppressor functions of C/EBPb: We previously found that C/EBPb is required for RasV12-induced cellular senescence in MEFs and showed that C/EBPb over-expression alone induces senescent-like growth arrest by a mechanism requiring RB:E2F. Cells lacking the tumor suppressor p19ARF bypass OIS and are transformed by Ras. We found that ARF, but not p53, is required to maintain C/EBPb levels in Ras-expressing fibroblasts, indicating that C/EBPb is part of an ARF-dependent tumor suppressor network. C/EBPb expression requires the Egr-1 and 2 transcription factors and activity of GSK3 kinases. Oncogenic Ras signaling inhibits GSK3 and decreases Egr levels, which at least partly explains down-regulation of C/EBPb in Ras-transformed cells. We are continuing to investigate how loss of ARF alters cellular responses to Ras and regulates Cebpb gene silencing. We are also using gene expression profiling and functional RNAi screens to identify target genes that mediate the cytostatic effects of C/EBPb. Regulation of C/EBPb activity by heterodimerization with C/EBPgamma: C/EBPg is a ubiquitously expressed protein that preferentially heterodimerizes with C/EBPb and other C/EBP family members. However, its biological functions are not well understood. We found that C/EBPg-deficient MEFs have reduced proliferative potential;this is in part due to the ability of C/EBPg to suppress the growth-inhibitory effects of C/EBPb by forming heterodimers. C/EBPg knockout cells display elevated levels of cyclin-dependent kinase (Cdk) inhibitors such as p15, p18, and p27, which may account for the decreased proliferative potential. C/EBPg null mice were originally reported to be nonviable;however, we found that the mutant mice survive when crossed onto a mixed strain background. Therefore, current work focuses on characterizing the phenotypes of adult C/EBPg deficient mice and investigating the role of C/EBPg in cell proliferation and tumorigenesis in vivo. Since C/EBPg alters the biological activity of C/EBPb, we are interested in identifying genomic targets of C/EBPb homo- and heterodimers. To that end we are developing a novel protein complementation/affinity tag (PCAT) method to enrich for chromosomal targets of various C/EBPb dimers. In principle, this method can be used to determine the interactome for any dimeric DNA-binding protein.